The present invention relates to a newly identified human carboxypeptidase. The invention also relates to polynucleotides encoding the carboxypeptidase. The invention further relates to methods using the carboxypeptidase polypeptides and polynucleotides as a target for diagnosis and treatment in carboxypeptidase-related disorders. The invention further relates to drug-screening methods using the carboxypeptidase polypeptides and polynucleotides to identify agonists and antagonists for diagnosis and treatment. The invention further encompasses agonists and antagonists based on the carboxypeptidase polypeptides and polynucleotides. The invention further relates to procedures for producing the carboxypeptidase polypeptides and polynucleotides.
Proteolytic enzymes are involved in many cellular processes. The carboxypeptidase family of enzymes catalyze, the cleavage of C-terminal amino acids of peptides and proteins, altering their biological activity. Lysosomal carboxypeptidase enzymes are highly concentrated in lysosomes, but may also be active extracellularly after their release from lysosomes in soluble form or bound to transmembrane or other membrane-associated proteins. Carboxypeptidases may cleave peptides in a sequence-specific manner. For example, prolylcarboxypeptidases cleave only peptides linked to proline residues (for example, des-Arg9-bradykinin, angiotensin II). There is also evidence that these enzymes are involved in terminating signal transduction by inactivating peptide ligands after receptor endocytosis.
In contrast to endoproteases which cleave internal peptide bonds of proteins and polypeptides, carboxypeptidases (CPs) catalyze the cleavage of only the C-terminal peptide bond, releasing one amino acid at a time. The two main groups of CPs include serine CPs and metallo-CPs, the serine CPs containing a signature trio of Ser, Asp, His in the active site. This trio is also contained in prolylendopeptidase serine proteases. Serine CPs include polycarboxypeptidase (PRCP) also referred to as angiotensinase C; and deamidase, also referred to as cathepsin A and lysosomal protective protein. See Skidgel et al. (1998) Immunological Reviews 161:129-141.
Metallo-CPs contain a signature glutamic acid as the primary catalytic residue and require zinc-binding for activity. Metallo-CPs can be grouped by substrate specificity into CPA and CPB types; the CPA type preferentially cleaving C-terminal hydrophobic residues, and the CPB type cleaving only peptides with C-terminal basic Arg or Lys residues. See R. A. Skidgel (1993) In: Hooper N M, ed. Zinc Metalloproteases in Health and Disease, London: Taylor and Francis, Ltd., p. 241-283.
CPM is a B type carboxypeptidase which is anchored on cell membranes via gylcosylphosphatidylinositol (GPI) association with its mildly hydrophobic stretch of 15 C-terminal amino acids. As in many other proteins sharing this anchoring mechanism, CPM is released from the membrane by bacterial phosphatidylinositol-specific phospholipase C. Human CPM is a glycoprotein of 426 amino acid residues with 43% identity to human intracellular secretory granular CP (CPE), 41% with the active 50 kDa subunit of human plasma CPN, and 15% with bovine pancreatic CPA or CPB. The active sites of these CPs contain conserved amino acid residues corresponding to the zinc binding residues His66Glu69 and His173, substrate binding residues Arg137 and Tyr242, and the catalytic Glu264, as designated for CPM. Sequence homologies around these conserved residues is high, with an identity between CPs M, E and N of approximately 70-90%. See Tan et al. (1989) J. Biol. Chem. 264:13165-13170; Deddish et al. (1990) J. Biol. Chem. 265:15083-15089; R. A. Skidgel (1993) In: Hooper N M, ed. Zinc Metalloproteases in Health and Disease, London: Taylor and Francis, Ltd., p. 241-283. CPM has been mapped to the chromosomal location of chromosome 12q13-q15 which is associated with a variety of solid tumors.
The optimal pH range of CPM is in the neutral range of 6.5-7.5. As no endogenous inhibitors are known for CPM, the enzyme is considered to be constitutively active. Synthetic inhibitors including Arg analogs DL-2mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA) and guanidinoethylmercaptosuccinic acid (GEMSA) inhibit CPM. See R. A. Skidgel (1991) In: Conn P M, ed. Methods in Neurosciences: Peptide Technology Vol. 6, Orlando: Academic Press, p. 373-385; Plummer et al. (1981) Biochem. Biophys. Res. Comm. 98: 448-254.
As with other B type regulatory CPs, CPM cleaves only C-terminal Arg or Lys residues; however, CPM has a preference for the C-terminal Arg. The penultimate amino acid also affects the rate of hydrolysis. Naturally occurring peptide substrates of CPM include bradykinin, Arg6- and Lys6 enkephalins, dynorphin A1-13 and epidermal growth factor (EGF). See Sidgel et al. (1989) J. Biol. Chem. 264:2236-2241; McGwire et al. (1995) J. Biol. Chem. 270:17154-17158.
CPM is primarily found on the plasma membrane, with highest levels found in lung and placenta. It is also present in kidney, blood vessels, intestine, brain and peripheral nerves. See R. A. Skidgel (1988) Trends Pharm. Sci. 9:299-304; Skidgel et al. (1984) Biochem. Pharmacol. 33: 3471-3478; Skidgel et al. (1991) FASEB J. 5: 1578; Nagae et al. (1992) J. Neurochem. 59:2201-2212; Nagae et al. (1993) Am. J. Respir. Cell Mol. Biol. 9:221-229. Expression of CPM is responsive to differentiation of monocytes and lymphocytes. See de Saint-Vis et al. (1995) Blood 86:1098-1105; Rehli et al. (1995) J. Biol. Chem. 270:15644-15649.
CPM participates in the control of peptide hormone activity at the cell surface and degradation of extracellular proteins and peptides. It catalyzes the second step in prohormone processing and removes C-terminal Arg or Lys residues from peptides released from prohormones. CPM functions as a soluble enzyme after its release from the plasma membrane and may function in the plasma membrane form to control peptide receptor activities. CPM can regulate receptor specificity of kinins by cleaving the C-terminal ARG9, for example, from bradykinin. The intact bradykinin binds the B2 receptor. The cleaved bradykinin (des-ARG9-bradykinin). Des-ARG9-bradykinin also binds the B1 receptors: stimulates IL-1 and tumor necrosis factor release from macrophages. Regulation of the B1 receptor is associated with injury or inflammation. CPM may also be involved with other inflammatory mediators, such as anaphylatoxin C5a which mediates histamine release. In addition, CPM may metabolize growth factors containing terminal Arg or Lys, such as EGF, EGF-like peptides, nerve growth factor (NGF) amphiregulin, hepatocyte growth factor, erythropoietin, and macrophage-stimulating protein. In the lung, varying levels of CPM are associated with pneumocystic or bacterial pneumonia or lung cancer, and in the placenta, CPM may protect the fetus from maternally derived peptides. See R. A. Skidgel (1992) J. Cardiovasc. Pharmacol. 20(Suppl. 9):S4-S9; Bhoola et al. (1992) Pharmacol. Rev. 44:1-80; R. A. Skidgel (1993) In: Hooper N M, ed. Zinc Metalloproteases in Health and Disease, London: Taylor and Francis, Ltd., p. 241-283; Dragovic et al. (1995) Am. J. Respir. Crit. Care Med. 152:760-764; Nagae et al. (1992) J. Neurochem. 59:2201-2212; MacFadden et al. (1988) FASEB J. 2:1179 (Abstract).
Another B-type regulatory CP metalloprotein is CPD, a membrane-bound glycoprotein. Human CPD is a protein of 1,377 amino acids with 75% identity with duck GP180 and 90% identity with rat CPD. Human CPD contains two hydrophobic regions located at the C- and N-termini. A 55-60 residue cytoplasmic domain is highly conserved among duck, human and rat sequences and may be significant in intracellular sorting, protein-protein interactions or endocytosis. CPD contains three tandem CP homology domains numbered sequentially from the N- to the C-terminus, and thereby may contain more than one active site. See Tan et al. (1997) Biochem. J. 327:81-87; Skidgel et al. (1993) In: Robertson J L S, Nicholls M G, eds. The Renin Angiotensin System, Vol. 1, London: Gower Medical Publishing, p. 10.1-10.10. CPD is located on human chromosome 17, 17P, 11.1-17q, 11.2.
CPD is primarily found on intracellular membranes, mainly in the Golgi, with some CPD found on the plasma membrane. The tissue distribution of CPD is wide and includes most duck tissues and mammalian tissues as well, including brain, pituitary, placenta, pancreas, adrenal, kidney, lung, heart, spleen, intestine, ovary, and testes. See McGwire et al. (1997) Life Sci. 60:715-724; Song et al. (1995) J. Biol. Chem. 270:25007-25013; Xin et al. (1997) DNA Cell Biol. 16:897-909; Tan et al. (1997) Biochem. J. 327:81-87; Song et al. (1996) J. Biol. Chem. 271:28884-28889.
The function of CPD is speculated to include peptide and protein processing in the constitutive secretory pathway after endoprotease cleavage of precursor proteins. The enzyme has an acidic pH optimum. Mammalian CPD may act as a hepatitis B virus binding protein, similar to the duck CPD. See R. A. Skidgel (1998) Immunological Reviews 161:129-141.
Serine CPs include PRCP and deamidase. PRCP cloned from a human kidney library indicates a glycoprotein of 51kDa3; and containing 496 amino acids, including a 30 residue signal peptide and a 15 residue propeptide. See Tan et al. (1993) J. Biol. Chem. 268:16631-16638. A serine repeat is found in the C-terminal half, similar to the serine repeat of a yeast CP encoded by the KEX1 gene.
PRCP has an acidic pH optimum for synthetic peptide substrates, but retains activity at neutral ranges with longer naturally occurring peptides. PRCP cleaves peptides only if the penultimate residue is proline. The enzyme does not cleave Pro-Pro-COOH or (OH)-Pro-Pro-COOH bond. See Odya et al. (1978) J. Biol. Chem. 253:5927-5931. Substrates of PRCP include des-Arg9-bradykinin and angiotensin II.
PRCP may be involved in terminating signal transduction by inactivating peptide ligands after receptor endocytosis. PRCP is contained in lysosomes and released in response to stimulation. The enzyme is widely distributed and found in human placenta, lung, liver, and kidney.
Another serine CP, deamidase, is likely a 94 kDa homodimer of 52 kDa subunits. Human platelet deamidase is activated by cleavage of a 14 amino acid fragment from the C-terminus. The enzyme binds and maintains activity and stability of -galactocidase and neuraminidase in lysosomes, a defect of which is associated with severe galactosialidosis. See Bonten et al. (1995) J. Biol. Chem. 270:26441-26445; Galjart et al. (1988) Cell 54:755-764; D""Azzo et al. (1982) Proc. Natl. Acad. Sci. 79:4535-4539. The gene for the human deamidase is mapped to chromosome 20 at q13.1.
Deamidase cleaves various peptides containing C-terminal or penultimate hydrophobic residues including substance P, angiotensin I, bradykinin, endothelin, and fMet-Leu-Phe. Like PRCP, deamidase is also found in lysosomes, and distributed in human placenta, lung, liver, and kidney. Like PRCP, deamidase is implicated in blocking part of the signal transduction pathway stimulated by peptides. Bradykinin, containing a C-terminal Arg9 and a penultimate hydrophobic amino acid Phe8, is cleaved by deamidase. Similarly, angiotensin, containing a C-terminal His and a penultimate Phe, is cleaved by deamidase. Accordingly, deamidase is implicated in termination of bradykinin activity on the B2 receptor to generate a B1 receptor agonist. Deamidase may also have a role in chemotaxis and in metabolism of the anti-cancer growth factor antagonist. See Skidgel et al. (1998) Immunological Reviews 161:129-141; Jackman et al. (1990) J Biol. Chem. 265:11265-11272; Jackman et al. (1995) Am. J. Respir. Cell Mol. Biol. 13:196-204; Hinek et al. (1996) Biol. Chem. 377:471-480; Jones et al. (1995) Peptides 16:777-783; Cummings et al. (1995) Biochem Pharmacol. 49:1709-1712.
Given the wide distribution and various physiological and pathological roles of carboxypeptidases, methods and compositions directed at regulating levels of these enzymes are useful for regulating peptide hormone activity, modulating metabolism of substance P, angiotensin I, angiotensin II, bradykinin, and endothelin, and regulation of signal transduction by inactivation of peptide ligands subsequent to receptor endocytosis.
Accordingly, carboxypeptidases are a major target for drug action and development. Therefore, it is valuable to the field of pharmaceuticals development to identify and characterize previously unknown carboxypeptidases. The present invention advances the state of the art by providing a previously unidentified human carboxypeptidase.
It is an object of the invention to identify novel carboxypeptidases.
It is a further object of the invention to provide novel carboxypeptidase polypeptides that are useful as reagents or targets in carboxypeptidase assays applicable to treatment and diagnosis of carboxypeptidase-related disorders.
It is a further object of the invention to provide polynucleotides corresponding to the novel carboxypeptidase polypeptides that are useful as targets and reagents in carboxypeptidase assays applicable to treatment and diagnosis of carboxypeptidase-related disorders and useful for producing novel carboxypeptidase polypeptides by recombinant methods.
A specific object of the invention is to identify compounds that act as agonists and antagonists and modulate the expression of the novel carboxypeptidase.
A further specific object of the invention is to provide compounds that modulate expression of the carboxypeptidase for treatment and diagnosis of carboxypeptidase-related disorders.
The invention is thus based on the identification of a novel human carboxypeptidase. The amino acid sequence is shown in SEQ ID NO 1. The nucleotide sequence is shown as SEQ ID NO 2.
The invention provides isolated carboxypeptidase polypeptides, including a polypeptide having the amino acid sequence shown in SEQ ID NO 1 or the amino acid sequence encoded by the cDNA deposited as ATCC No. PTA-1643 on Apr. 5, 2000 (xe2x80x9cthe deposited cDNAxe2x80x9d).
The invention also provides isolated carboxypeptidase nucleic acid molecules having the sequence shown in SEQ ID NO 2 or in the deposited cDNA.
The invention also provides variant polypeptides having an amino acid sequence that is substantially homologous to the amino acid sequence shown in SEQ ID NO 1 or encoded by the deposited cDNA.
The invention also provides variant nucleic acid sequences that are substantially homologous to the nucleotide sequence shown in SEQ ID NO 2 or in the deposited cDNA.
The invention also provides fragments of the polypeptide shown in SEQ ID NO 1 and nucleotide sequence shown in SEQ ID NO 2, as well as substantially homologous fragments of the polypeptide or nucleic acid.
The invention further provides nucleic acid constructs comprising the nucleic acid molecules described herein. In a preferred embodiment, the nucleic acid molecules of the invention are operatively linked to a regulatory sequence.
The invention also provides vectors and host cells for expressing the carboxypeptidase nucleic acid molecules and polypeptides, and particularly recombinant vectors and host cells.
The invention also provides methods of making the vectors and host cells and methods for using them to produce the carboxypeptidase nucleic acid molecules and polypeptides.
The invention also provides antibodies or antigen-binding fragments thereof that selectively bind the carboxypeptidase polypeptides and fragments.
The invention also provides methods of screening for compounds that modulate expression or activity of the carboxypeptidase polypeptides or nucleic acid (RNA or DNA).
The invention also provides a process for modulating carboxypeptidase polypeptide or nucleic acid expression or activity, especially using the screened compounds. Modulation may be used to treat conditions related to aberrant activity or expression of the carboxypeptidase polypeptides or nucleic acids.
The invention also provides assays for determining the activity of or the presence or absence of the carboxypeptidase polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.
The invention also provides assays for determining the presence of a mutation in the polypeptides or nucleic acid molecules, including for disease diagnosis.
In still a further embodiment, the invention provides a computer readable means containing the nucleotide and/or amino acid sequences of the nucleic acids and polypeptides of the invention, respectively.